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Chondroitin sulfate (CS) of various molecular weight (MW), up to ∼3 kDa, were produced and tested for uronic acid carbazole assay and cetylpyridinium chloride (CPC) titration showing an evident decrease in the assays depending on the CS MW. The described results for uronic acid assay by carbazole reaction and CPC titration of CS poses the problem to know the MW values before their application and to use comparable standards to obtain reliable results. Otherwise, the related quantitative data can be affected by a great error and fake certificate of analysis., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Dried blood spot (DBS) technology is a cheap and easy method largely applied in newborn screening. Mucopolysaccharidoses (MPS) are characterized by the deficit of enzymes that degrade glycosaminoglycans (GAGs) characterized by progressive worsening of the conditions. For a possible early diagnosis of MPS, we developed a method of uronic acid (UA)-GAGs determination in DBS of 600 healthy newborns and from a small group of MPS subjects matched for age. Spotted blood UA-GAGs of the normal newborns are composed of 67.2% chondroitin sulfate (CS), 28.6% heparan sulfate (HS) and 4.4% hyaluronic acid with a CS/HS ratio of 2.35 and a total GAGs content of 0.43 μg/DBS. A chemical evaluation of CS and HS structure was performed by measuring their disaccharide composition, sulfation and the overall charge density. The DBS of four different MPS types presented an increase of total or single UA-GAGs content and/or modifications of the CS and HS disaccharide composition as well as chemical signature also related to the MPS enzymatic defect. The modifications of the UA-GAGs composition, parameters and structure of healthy newborns determined in DBS would be useful for a possible early diagnosis of various MPS types., (Copyright © 2018. Published by Elsevier Inc.)

Keratan sulfate (KS) is present as a contaminant in chondroitin sulfate (CS) mainly extracted from shark cartilage. We report a selective removal procedure of KS in CS samples by means of sequential precipitation with ethanol. Purified shark CS containing approximately 10% to 15% KS was subjected to a precipitation procedure in the presence of increasing percentages of saturated ethanol. In contrast to other solvents, 1.0 volume of ethanol was able to selectively purify CS, with a purity of approximately 100%, from KS. The current selective and simple procedure appears to be a reliable industrial preparation of CS devoid of large amounts of the residual KS., (Copyright © 2013 Elsevier Inc. All rights reserved.)

A high-resolution normal-phase high-performance liquid chromatography-fluorescence detection-electrospray ionization-mass spectrometry separation and structural characterization of the main oligosaccharides along with lactose from human milk samples is described. A total of 22 commercially available oligosaccharides were fluorotagged with 2-aminoacridone and separated on an amide column and identified on the basis of their retention times and mass spectra. Derivatized species having mass lower than approximately 800 to 900 exhibited mainly [M-H](-1) anions, oligomers with mass up to approximately 1000 to 1100 were represented by both [M-H](-1) and [M-2H](-2) anions, and oligomers greater than approximately 1200 to 1300 were characterized by a charge state of -3. Furthermore, the retention times were directly related to the glycans' molecular mass. Human milk samples from the four groups of donors (Se±/Le±) were analyzed for their composition and amount of free oligosaccharides after rapid and simple prepurification and derivatization steps also in the presence of lactose in high content. This analytical approach enabled us to perform the determination of species not detected by traditional techniques, such as sialic acid, as well as of species present in low content easily mistaken with other peaks. Finally, labeled human milk oligosaccharides were analyzed without any interference from excess fluorophore or interference from proteins, peptides, salts, and other impurities normally present in this complex biological fluid., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Mucopolysaccharidoses (MPS) diagnosis is often delayed and irreversible organ damage can occur, making possible therapies less effective. This highlights the importance of early and accurate diagnosis. A high-throughput procedure for the simultaneous determination of glucosamine and galactosamine produced from urinary galactosaminoglycans and glucosaminoglycans by capillary electrophoresis (CE) and HPLC has been performed and validated in subjects affected by various MPS including their mild and severe forms, Hurler and Hurler-Scheie, Hunter, Sanfilippo, Morquio, and Maroteaux-Lamy. Contrary to other analytical approaches, the present single analytical procedure, which is able to measure total abnormal amounts of urinary GAGs, high molecular mass, and related fragments, as well as specific hexosamines belonging to a group of GAGs, would be useful for possible application in their early diagnosis. After a rapid urine pretreatment, free hexosamines are generated by acidic hydrolysis, derivatized with 2-aminobenzoic acid and separated by CE/UV in ∼10min and reverse-phase (RP)-HPLC in fluorescence in ∼21min. The total content of hexosamines was found to be indicative of abnormal urinary excretion of GAGs in patients compared to the controls, and the galactosamine/glucosamine ratio was observed to be related to specific MPS syndromes in regard to both their mild and severe forms. As a consequence, important correlations between analytical response and clinical diagnosis and the severity of the disorders were observed. Furthermore, we can assume that the severity of the syndrome may be ascribed to the quantity of total GAGs, as high-molecular-mass polymers and fragments, accumulated in cells and directly excreted in the urine. Finally, due to the high-throughput nature of this approach and to the equipment commonly available in laboratories, this method is suitable for newborn screening in preventive public health programs for early detection of MPS disorders, diagnosis, and their treatment., (Copyright © 2010 Elsevier Inc. All rights reserved.)

In this study, we developed an on-line reverse-phase high-performance liquid chromatography-electrospray ionization-mass spectrometry (RP-HPLC-ESI-MS) separation and structural characterization of hyaluronan (HA)/chondroitin sulfate (CS)/dermatan sulfate (DS) disaccharides released by enzymatic treatment and derivatized with 2-aminoacridone (AMAC), providing a high-resolution system also applicable by using a further fluorimetric detector (Fp) before ESI-MS spectral acquisition. Isomeric nonsulfated HA and CS/DS disaccharides, isomeric monosulfated and isomeric disulfated CS/DS disaccharides, and the trisulfated species were distinctly separated and unambiguously identified by their retention times and mass spectra in negative ionization mode. In general, no multiply charged ions were detected even for highly charged disaccharides, but the presence of desulfonated products for highly sulfated species due to the relative instability of sulfo groups was observed. RP-HPLC-ESI-MS of each AMAC disaccharide was found to be linear from 3 to 500 ng with very high coefficient of correlation values due to the high efficiency of separation and the sharp outline of the peaks. Various CS/DS samples were characterized for disaccharide composition, and minor oligomer species identified as GalNAcSO(4) at the nonreducing end of chains was observed as a common component of these macromolecules. Furthermore, purified endogenous normal human plasma CS disaccharides were also evaluated by means of RP-HPLC-(Fp)-ESI-MS., (Copyright 2009 Elsevier Inc. All rights reserved.)

A simple, accurate, and robust quantitative capillary electrophoresis (CE) method for the determination of oversulfated chondroitin sulfate (OSCS) as a contaminant in heparin (Hep) preparations is described. After degradation of the polysaccharides by acidic hydrolysis, the hexosamines produced (i.e., GlcN from Hep and GalN from OSCS) were derivatized with anthranilic acid (AA) and separated by means of CE in approximately 10 min with high sensitivity detection at 214 nm (limit of detection [LOD] of approximately 200 pg). Furthermore, AA-derivatized GlcN and GalN showed quite similar molar absorptivity, allowing direct and simple quantification of OSCS in Hep samples. Moreover, a preliminary step of specific enzymatic treatment by using chondroitin ABC lyase may be applied for the specific elimination of interference in the analysis due to the possible presence in Hep samples of natural chondroitin sulfate and dermatan sulfate impurities, making this analytical approach highly specific for OSCS contamination given that chondroitin ABC lyase is unable to act on this semisynthetic polymer. The CE method was validated for specificity, linearity, accuracy, precision, LOD, and limit of quantification (LOQ). Due to the very high sensitivity of CE, as little as 1% OSCS contaminant in Hep sample could be detected and quantified. Finally, a contaminated raw Hep sample was found to contain 38.9% OSCS, whereas a formulated contaminated Hep was calculated to have 39.7% OSCS.

In this paper, we report a rapid, sensitive, and quantitative procedure to conduct disaccharide compositional analyses of keratan sulfates (KS) by means of high-performance liquid chromatography (HPLC) separation and postcolumn derivatization with 2-cyanoacetamide and fluorimetric detection of products generated by hydrolysis of this glycosaminoglycan with Bacillus sp. keratanase II or Escherichia freundii endo-beta-galactosidase. Following E. freundii endo-beta-galactosidase digestion of bovine corneal KS, the monosulfated disaccharide glcNAc6sbeta(1-->3)gal, accounting for approximately equals 95% nmol and 50% yield products, is produced. On the contrary, bovine corneal KS treated with endo-beta-N-acetylglucosaminidase (keratanase II) from Bacillus sp. generates two major products, the monosulfated disaccharide galbeta(1-->4)glcNAc6s ( approximately equals 50% nmol product) and the disulfated disaccharide gal6sbeta(1-->4)glcNAc6s ( approximately equals 40% nmol product) for over 90% nmol products. These disaccharides are separated and readily determined within 30 min by using a linear-gradient strong anion-exchange separation. A linear relationship was found for the two purified disaccharides over a wide range of concentrations, from approximately equals 108 pmol, 50 ng, to 2,160 pmol, 1,000 ng, for the disaccharide galbeta(1-->4)glcNAc6s, and from 92 pmol, 50 ng, to 1,840 pmol, 1,000 ng, for the disaccharide gal6sbeta(1-->4)glcNAc6s. HPLC analysis was applied to the quantitative and qualitative determination of KS produced by 3T3-J2 murine fibroblasts in the cell medium. The amount of KS was found to be 2.80+/-0.34 microg/ml/10(6) cells and composed of approximately equals 71% nmol of disaccharide galbeta(1-->4)glcNAc6s and 18% nmol of the disulfated disaccharide gal6sbeta(1-->4)glcNAc6s having approximately equals 1.20 sulfate groups/disaccharide. Our data illustrate that the HPLC procedure reported represents an improved approach for the quantitative and compositional microanalyses of KS, especially applicable to experimentation involving small amounts ( approximately 50 ng) of this glycosaminoglycan and in relation to its biological function and pathological importance.

A sensitive agarose-gel electrophoresis method has been developed for the visualization of lipopolysaccharide (LPS) samples from several Escherichia coli serotypes, such as 026:B6, 055:B5, 0128:B12, 0111:B4, 0127:B8, and K235. This method can detect as little as 0.5-6 microg of LPS depending on the serotype by treatment of the plates with toluidine blue, and it is able to measure sub-microgram amounts of samples, approx. 0.05-0.5 microg, when the staining with toluidine blue followed by Stains-All procedure is adopted. Treatment of LPS with alkali under anhydrous conditions removes the ester-linked fatty acids and the phosphate groups of the Lipid A component, producing the detoxified LPS. The carbohydrate neutral moiety obtained from the hydrolysate does not migrate during electrophoresis. This was utilized to monitor quantitatively the removal process of the Lipid A component for the formation of the detoxified LPS.

A system capable of resolving all the known unsaturated nonsulfated, mono- and disulfated disaccharides derived from chondroitin sulfate samples, dermatan sulfate, and hyaluronic acid after their derivatization with dansylhydrazine and separation by HPLC and fluorimetric detection is reported. This method was found superior to others in that unsaturated disaccharides can be separated with good resolution in about 50 min in an isocratic solvent with a sensitivity greater than about 50 pmol (approx 20-30 ng) and linearity from 50 to 500 pmol. The system was applied to the analysis of various chondroitin sulfate samples, including highly sulfated species and dermatan sulfate, and also to a defructosylated polysaccharide with a chondroitin backbone purified from Escherichia coli U1-41. Excellent agreement was obtained with traditional compositional analysis performed by anion-exchange HPLC separation and UV absorption at 230 nm., (Copyright 2000 Academic Press.)

The separation of sulfated glycosaminoglycans in mixtures by agarose-gel electrophoresis and the recovery of single polysaccharide bands has been applied to the characterization of polysaccharides extracted from tissues without previous purification of single species. Sulfated glycosaminoglycans, heparin with its two components, slow-moving and fast-moving, heparan sulfate, dermatan sulfate, and chondroitin sulfate, were separated to microgram level by conventional agarose-gel electrophoresis. After their separation, they were fixed in the agarose-gel matrix by precipitation in a cetyltrimethylammonium bromide solution, making them visible on a dark background. After recovery of gel containing the fixed bands, high temperatures (90 degrees C for 15 min) were necessary to dissolve the gel matrix, and a solution of NaCl (3 M) was used to release sulfated polysaccharides from the complex with cetyltrimethylammonium. After precipitation of glycosaminoglycans in the presence of ethanol, the recovery of slow-moving heparin, fast-moving heparin, heparan sulfate, dermatan sulfate, and chondroitin sulfate was from 1 to 10 microg, with a percentage greater than 45% and a purity above 90%. Sulfated glycosaminoglycans in mixtures recovered from gel matrix as single species were evaluated for purity and characterized for unsaturated disaccharides after treatment with bacterial lyases (heparinases for heparin and heparan sulfate samples, and chondroitinases for dermatan sulfate and chondroitin sulfate) and molecular mass. Bovine lung and heart Glycosaminoglycans were extracted and separated into single species by agarose-gel electrophoresis and recovered from gel matrix after treatment in cetyltrimethylammonium solution. Unsaturated disaccharides pattern, the sulfate to carboxyl ratio, and the molecular mass of each single polysaccharide species were determined., (Copyright 1999 Academic Press.)

A rapid and simple electrophoretic separation method on nitrocellulose membranes by a single run of glycosaminoglycans, mainly slow-moving and fast-moving heparins, dermatan sulfate, and chondroitin sulfate, is reported. Different dyes, azure A, toluidine blue, and Alcian blue, were used to stain the nitrocellulose strips, and azure A showed the highest sensitivity. The mobility for slow-moving heparin was 0.00 cm/A/min, for fast-moving species about 2.87 x 10(-3) cm/A/min, for dermatan sulfate 4.00 x 10(-3) cm/A/min, and for chondroitin sulfate about 5.75 x 10(-3) cm/A/min. Calibration curves were determined by the increasing amounts (from 0. 1 to 2.5 microg) of single glycosaminoglycan species. The curves demonstrated a linear relationship between the amount of glycosaminoglycans (slow-moving and fast-moving heparin, dermatan sulfate, and chondroitin sulfate) and the optical density at 600 nm calculated by densitometric scanning. The limit of detection for fast-moving heparin, dermatan sulfate and chondroitin sulfate was about 0.2-0.3 microg, while that for slow-moving heparin was 0.1 microg. Hyaluronic acid, keratan sulfate, and highly sulfated chondroitin sulfate from shark cartilage were also separated electrophoretically on nitrocellulose. No difference in mobility was detected for chondroitin sulfates with different structure and physicochemical properties. Hyaluronic acid has about the same mobility as that of dermatan sulfate, while keratan sulfate has a mobility intermediate between those of dermatan sulfate and chondroitin sulfate (about 4.75 x 10(-3) cm/A/min). Glycosaminoglycans extracted and purified from bovine aorta and bovine lung were submitted to electrophoresis on nitrocellulose and the amount of each polysaccharide species was calculated by densitometric scanning. By this analysis bovine aorta glycosaminoglycans consist of about 11% fast-moving heparin (heparan sulfate), 23% dermatan sulfate, and 65% chondroitin sulfate. Bovine lung polysaccharides are composed of about 16% slow-moving and 46% fast-moving species (62% heparin), 15% dermatan sulfate, and 22% chondroitin sulfate.